Fluorophore coupled secondary antibodies

Teased sciatic nerves and frozen optic nerve sections were prepared as described previously (Amor et al., 2017 ). Slides (either tissues or cultured cells) were post fixed with cold methanol, washed with PBS and incubated with blocking solution (5% fish skin gelatin or normal goat serum and 0.1% Triton X-100, in PBS) for 1 hour at room temperature. Samples were incubated at 4°C with the mixture of primary antibodies diluted in blocking solution for 12 hours, washed with PBS, incubated for 45 minutes at ambient temperature with fluorophore-coupled secondary antibodies (Jackson Laboratories and Molecular Probes), and then washed with PBS and mounted with Elvanol. Fluorescence images were obtained either using Nikon eclipse E1000 microscope with a Hamamatsu ORCA-ER CCD camera, or using a Zeiss LSM700 confocal microscope. Images were acquired and processed using the Zen2012 software (Carl Zeiss). For electron microscopy, sciatic nerves were exposed and fixed with 4% PFA, 2.5% glutaraldehyde, and 0.1M sodium cacodylate pH 7.4 in PBS for 40 minutes. Nerves were then removed and incubated over-night in the same fixative. Samples processing was carried out as previously described (Novak et al., 2011 (link)). Sections were imaged using a Philips CM-12 transmission electron microscope.